Genes encoding proteins involved in post-translational modifications, particularly in glycan synthesis, comprise glycosyltransferases and sulfotransferases, and are essential for regulating cell communications and activities. Glycan synthesis and modification enzymes encoded by the LARGE, ST8SIA1 and HS6ST2 genes are resident of the Golgi apparatus.
Precisely, LARGE gene encodes the N-acetylglycosaminyl transferase that is involved in glycosylation of alpha-dystroglycan (α-DG) (Hewitt J E. Investigating the functions of LARGE: lessons from mutant mice. Methods Enzymol 2010: 479: 367-386, Hewitt J E. Abnormal glycosylation of dystroglycan in human genetic disease. Biochim Biophys Acta 2009: 1792: 853-861).
α-DG is a major non-integrin adhesion molecule expressed at the interface between the basement membrane and the cell membrane, which links cytoskeletal actin to extracellular matrix and is likely involved in adhesion and regulation of signalling pathways (Ibraghimov-Beskrovnaya O. Ervasti J M, Leveille C J, Slaughter C A, Sernett S W, Campbell K P. Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature 1992: 355: 696-702, Barresi R, Campbell K P. Dystroglycan: from biosynthesis to pathogenesis of human disease. J Cell Sci 2006: 119: 199-207). α-DG requires extensive posttranslational processing in order to function as an extracellular matrix receptor (Yoshida-Moriguchi T, Yu L, Stalnaker S H, et al. O-mannosyl phosphorylation of alpha-dystroglycan is required for laminin binding. Science 2010: 327: 88-92). α-DG has been implicated in several cell functions, such as growth control, cytoskeletal organization that may lead to cellular internal and external tension, cell polarization, differentiation and movement (Sgambato A, Caredda E, Leocata P, et al. Expression of alpha-dystroglycan correlates with tumour grade and predicts survival in oral squamous cell carcinoma. Pathology 2010: 42: 248-254).
α-DG is expressed in epithelial cells and essential for epithelial development. Deficiency of perlecan, a major ligand of α-DG, enhances shedding of α-DG, destabilizes epithelial DG complex, and makes it accessible to proteolytic processing (Herzog C, Has C, Franzke C W, et al. Dystroglycan in skin and cutaneous cells: beta-subunit is shed from the cell surface. J Invest Dermatol 2004: 122: 1372-13 80).
The human LARGE gene is required for the generation of functional α-DG and ligand binding activity of α-DG (Yoshida-Moriguchi T, Yu L, Stalnaker S H, et al. O-mannosyl phosphorylation of alpha-dystroglycan is required for laminin binding. Science 2010: 327: 88-92).
Abnormal glycosylation of α-DG disrupts binding to laminin and other proteins of the extracellular matrix resulting in diseases termed dystroglycanopathies (Moore C J, Goh H T, Hewitt J E. Genes required for functional glycosylation of dystroglycan are conserved in zebrafish. Genomics 2008: 92: 159-167; Martin P T. Congenital muscular dystrophies involving the O-mannose pathway. Curr Mol Med 2007: 7: 417-425; Moore C J, Hewitt J E. Dystroglycan glycosylation and muscular dystrophy. Glycoconj J 2009: 26: 349-357). The dystroglycanopathies show clinical phenotypes (in particular, muscular dystrophy, CNS abnormalities, and eye defects) that are presumed to be primarily due to this deficiency in α-DG glycosylation. Inactivating mutations of LARGE also reduce the functional glycosylation of α-DG and lead to dystroglycanopathies in mouse and humans. Mutations in the human LARGE gene are the basis of the dystroglycanopathy termed MDC1D (Longman C, Brockington M, Torelli S, et al. Mutations in the human LARGE gene cause MDC1D, a novel form of congenital muscular dystrophy with severe mental retardation and abnormal glycosylation of alpha-dystroglycan. Hum Mol Genet 2003: 12: 2853-2861). The mouse LARGE ortholog is 97.8% identical to human LARGE, and a deletion in mouse LARGE is the basis of the myd mouse that develops a muscular dystrophy similar to MDC1D.
Overexpression of LARGE in cultured fibroblasts from patients with different dystroglycanopathies generates functional α-DG. In mutant CHO cells in which transfer of galactose, fucose or sialic acid to glycoconjugates is compromised, LARGE was able to induce α-DG glycosylation. In cells carrying loss of function mutations in other genes in this pathway such as POMT1 or POMGnT1, overexpression of LARGE can induce glycosylation on α-DG. Upregulation of LARGE is considered as therapeutic strategy for dystroglycanopathies that can rescue α-DG glycosylation and function.
Like the LARGE gene, the ST8SIA1 gene encodes for a type II transmembrane protein involved in the synthesis of gangliosides. The HS6ST2 gene encodes an enzyme catalyzing the transfer of sulphate to heparan sulphate.
It is thus desirable and important to provide products or active agents which prevent, reduce or even inhibit the cellular senescence, particularly the keratinocyte senescence, more particularly UV-induced cell senescence.
The present invention thus provides a method for identifying such useful agents.